Thiophene-linked tetramethylbodipy-labeled nucleotide for viscosity-sensitive oligonucleotide probes of hybridization and protein-DNA interactions
Language English Country Great Britain, England Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
31919486
DOI
10.1039/c9ob02634g
Knihovny.cz E-resources
- MeSH
- DNA-Binding Proteins metabolism MeSH
- DNA-Directed DNA Polymerase metabolism MeSH
- DNA metabolism MeSH
- Spectrometry, Fluorescence MeSH
- Nucleic Acid Hybridization * MeSH
- Cations MeSH
- Humans MeSH
- Lipids chemistry MeSH
- Cell Line, Tumor MeSH
- Nucleotides chemical synthesis chemistry MeSH
- Oligonucleotide Probes metabolism MeSH
- Solvents chemistry MeSH
- Base Sequence MeSH
- Boron Compounds chemistry MeSH
- Temperature MeSH
- Thiophenes chemistry MeSH
- Protein Binding MeSH
- Viscosity MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene MeSH Browser
- DNA-Binding Proteins MeSH
- DNA-Directed DNA Polymerase MeSH
- DNA MeSH
- Cations MeSH
- Lipids MeSH
- Nucleotides MeSH
- Oligonucleotide Probes MeSH
- Solvents MeSH
- Boron Compounds MeSH
- Thiophenes MeSH
Cytosine 2'-deoxyribonucleoside dCTBdp and its triphosphate (dCTBdpTP) bearing tetramethylated thiophene-bodipy fluorophore attached at position 5 were designed and synthesized. The green fluorescent nucleoside dCTBdp showed a perfect dependence of fluorescence lifetime on the viscosity. The modified triphosphate dCTBdpTP was substrate to several DNA polymerases and was used for in vitro enzymatic synthesis of labeled oligonucleotides (ONs) or DNA by primer extension. The labeled single-stranded ONs showed a significant decrease in mean fluorescence lifetime when hybridized to the complementary strand of DNA or RNA and were also sensitive to mismatches. The labeled dsDNA sensed protein binding (p53), which resulted in the increase of its fluorescence lifetime. The triphosphate dCTBdpTP was transported to live cells where its interactions could be detected by FLIM but it did not show incorporation to genomic DNA in cellulo.
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